Genetic mutations that cause congenital heart malformations are often heterozygous and involve a partial reduction in protein dosage or an increase in protein activity. Although developmental events are precisely controlled by signaling pathways and transcriptional networks, our studies have revealed an intertwined layer of post-transcriptional regulation that involves microRNAs (miRNAs) that titrate protein dosage. The muscle-specific miRNA miR-1 is co-transcribed with a second miRNA, miR-133, and participates in septal formation, cell-cycle regulation, cardiac conduction, and other aspects of cardiac development and homeostasis. miR-1 and miR-133 are encoded in two loci as a result of a gene duplication, with identical mature sequences of miR-1-1 and miR-1-2, as well as miR-133a and miR- 133b. Loss of two redundant copies of miR-133 results in a ventricular septal defect, similar to deletion of miR-1-2. miR-1 and miR-133 appear to function in concert in some biological settings, but have opposing functions in others. The function of miR-1 is dose sensitive, as shown by gene targeting of miR-1-2, although the full function of miR-1 awaits compound deletion of miR-1-2 and its redundant allele, miR-1-1. Several targets of miR-1 are known, including Hand2, Irx5 and Delta-like 1. However, the contribution of individual targets to miR-1's function in vivo is unknown, and most targets of miR-1 and miR-133 are also unknown. We hypothesize that miR-1 is required for cardiac progenitor development in vivo and for postnatal cardiac function and that a discrete set of mRNA targets play major roles in mediating miR-1's function. We also hypothesize that miR-1 and miR-133 converge on common targets to cooperatively regulate cellular decisions but have other targets that mediate distinct functions. To test these hypotheses, we propose three specific aims: Aim 1) To determine the dose- dependent requirement of miR-1 in cardiac development and in post-natal cardiac function by analyzing compound deletions of miR-1-1 and miR-1-2; Aim 2) To determine whether repression of individual miR-1 targets mediates major functions of miR-1 in vivo; and Aim 3) To determine whether miR-1 and miR-133 share common targets upon which they can cooperate or synergize and whether they have distinct targets that mediate opposing functions. These studies will utilize several innovative approaches and will reveal miRNA and transcriptional networks that titrate protein dosage to control critical events in cardiogenesis. PUBLIC HEALTH RELEVANCE: Quantitative disruption of the molecular networks regulating cardiac development underlies many forms of congenital heart disease. In this proposal, we integrate the precise control of protein dosage through small RNAs, known as microRNAs, with other known regulators of cardiogenesis to understand the multiple levels by which heart formation is controlled.